Ketoreductases and uses thereof

Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds.

What is claimed is: 
     
       1. An isolated or engineered polypeptide having ketoreductase activity which comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2 and having an L at the amino acid residue corresponding to residue 147 of SEQ ID NO:2. 
     
     
       2. The polypeptide of  claim 1  in which the amino acid sequence further comprises one or more features selected from: residue corresponding to residue 94 is an aromatic amino acid or G; residue corresponding to residue 96 is any amino acid other than S/N; residue corresponding to residue 153 is an aliphatic amino acid residue other than L; residue corresponding to residue 199 is any amino acid residue other than L; residue corresponding to residue 202 is G or an aliphatic amino acid residue other than A; and residue corresponding to residue 206 is an aromatic amino acid residue. 
     
     
       3. The polypeptide of  claim 2  in which the amino acid sequence further comprises one or more of features selected from: residue corresponding to residue 94 is F, W, H, Y; residue corresponding to residue 96 is G, I, C or an aromatic amino acid; residue corresponding to residue 153 is G or A; residue corresponding to residue 199 is K, I, N, R, V, Q, or W; residue corresponding to residue 202 is I, L, or G; and residue corresponding to residue 206 is F. 
     
     
       4. The polypeptide of  claim 2  in which the amino acid sequence further comprises one or more of the features selected from: residue 49 is a polar amino acid residue other than K; residue 53 is an acidic amino acid residue; residue 54 is a small or aliphatic amino acid residue other than T/P; residue 60 is an aliphatic amino acid residue other than V; residue 95 is an aliphatic amino acid other than V; residue 97 is a small amino acid or G; residue 109 is a basic amino acid residue other than K; residue 165 is a hydroxyl or small amino acid residue; residue 197 is a small amino acid residue or G; residue 223 is an aliphatic amino acid residue other than L; and residue 233 is a small amino acid residue or G. 
     
     
       5. The polypeptide of  claim 4  in which the amino acid sequence comprises one or more features selected from: residue 49 is R; residue 53 is D; residue 54 is A; residue 60 is A; residue 95 is L; residue 97 is G; residue 109 is R; residue 165 is T; residue 197 is G; residue 223 is V; and residue 233 is G. 
     
     
       6. The polypeptide of  claim 1  which is capable of converting acetophenone to (R)-1-phenylethanol. 
     
     
       7. The polypeptide of  claim 1  which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 110, 122, 124, 126, 136, and 138.